It is increasingly recognized that gut flora and gut barrier function play a pivotal role in the pathogenesis of several forms of liver disease including ALD. Gut-derived endotoxins normally penetrate the gut epithelium in only trace amounts due to tightly regulated intestinal barrier function. However, endotoxin leakiness is increased under pathological conditions, including alcohol abuse. The barrier function of intestinal epithelium Is provided in part by paracellular apical junction complexes, including tight and adherens junctions, and by a mucus layer. In our initial systems biology approach to ALD, we found that intestinal trefoil factor (ITF) was by far the most altered (~50-fold down regulated) protein in the liver following chronic alcohol feeding in rats, and subsequent studies also showed marked down regulation of ITF mRNA and protein in the intestines of alcohol fed mice. ITF plays a critical role in the formation and stabilization of intestinal mucus barrier and enhancement of rapid intestinal repair restitution. Formation and restitution of the barrier is also dependent on uninterrupted mucosal blood flow. Intestinal epithelial cells are positioned between an anaerobic lumen and a highly metabolic lamina propria forming a steep physiologic oxygen gradient. Increased hypoxia- inducible factor (HIF) signaling represents a major adaptive response to hypoxic stress by upregulating a variety of gene products, such as ITF and mucins, to form an integrated epithelial barrier. Our preliminary studies demonstrated that chronic alcohol administration decreases intestinal HIF-2?, ITF and tight junction protein expression resulting in increased endotoxemia and ultimately liver injury. Moreover, supplementation with a probiotic strain, Lactobacillus rhamnosus GG (LGG), increased intestinal HIF-2? and ITF expression, decreased endotoxemia and attenuated alcohol induced liver injury. We hypothesize that alcohol exposure causes detrimental metabolic changes favoring intestinal oxidative stress, inflammation, and alterations of HIF responsive signaling with decreased ITF, leading to intestinal barrier dysfunction, blood endotoxemia and ALD. We postulate that probiotic supplementation will attenuate alcohol-induced intestine and liver injury, in part by increasing HIF-mediated expression of mucus protective factors such as ITF in the intestine. We test our hypothesis with 4 specific aims using in vitro, animal, and human studies approaches. Our ultimate goal is the development of new therapies for ALD. RELEVANCE (See instructions): Alcohol overuse/abuse induces organ injury in multiple organs. This translational research project will examine the mechanisms of the beneficial effects of probiotics on the intestine and the liver in an experimental mouse model of alcoholic liver disease (ALD) and in mild human alcoholic hepatitis (AH).